PHSI3009 Frontiers in Cellular Physiology 2017

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1 Overview of PHSI3009 L2 Cell membrane and Principles of cell communication L3 Signalling via G protein-coupled receptor L4 Calcium Signalling L5 Signalling via Growth Factors L6 Signalling via small G-protein and MAPK L7 Mechanisms for Insulin Secretion L8 Control of Blood Glucose L9/10 Trafficking: Exocytosis and Endocytosis L11 Epithelial Na + Channel, ENaC L12 Regulation of ENaC Expression 1 L13 Regulation of ENaC Expression 2 L14 CFTR and Cystic Fibrosis L15 Cholera and Fluid Secretion L17 K + channels L18 ATP-dependent and Ca 2+ -dependent K + channels L19 Ca 2+ channels and disease L20 Maximising Transport Efficiency L21 Anion transporters and disease L22 Organic ion transporters and disease 1

2 Lecture 2 (Cell membrane and Principles of cell communication) Understand functions of the cell membrane Describe principles of membrane transport Understand electrical properties of the cell membrane and the role of ion channels and transporters Understand basic process of cell communication Understand how cell surface receptors relay signals Understand functional roles of intracellular signalling complex The cell membrane Separates interior of cell from environment selective permeability Internal compartmentalisation of eukaryotic cells Provide space for biochemical reactions Phospholipid bilayer contain: o Protein channels o Integral membrane protein o Peripheral membrane protein o Glycoprotein and glycolipid Most integral membrane proteins are transmembrane proteins transmembrane domain Perform specific tasks o Can be restricted to specific domains (i.e. apical/lateral/basal) Function as large complexes o Receptors, membrane tp, e- tp and oxidative phosphorylation o Control of interaction between cells cell-to-cell communication Transporters in epithelial cells are asymmetrically distributed I.e. CFTR and bicarbonate transporter are expressed only on apical surfaces Principle of membrane transport Simple diffusion = direct tp across lipid bilayer o Small non-charge and non-polar molecules Transporter-mediated (PASSIVE and ACTIVE) o Carriers o Ion channels Endocytosis + exocytosis o Reserved for larger molecules PASSIVE transport Depend on selectivity and moves down the concentration gradient [High Low] Simple diffusion Facilitated diffusion o Molecule bind to carrier protein in membrane and is carried across to the other side ACTIVE transport Require ATP to work AGAINST the concentration gradient [Low High] Carrier-mediated active transport o I.e. Na+/K+ ATPase Endocytosis 2

3 Mechanisms of transporters (PASSIVE or ACTIVE) 1. Binding of solute 2. Reversible conformational change 3. Expose solute to another side of the membrane Na+/glucose co-transporter (SGLT) Due to the asymmetric distribution of transporters (in this case, SGLT on the apical surface only) Allow transcellular glucose transport Ion channels and selective filter Ions move down their electrochemical gradient, w/o ATP input Selectivity filter = selective for specific species of ion 3

4 Cell communication Contact-dependent o Membrane-bound signal molecule receptor on target cell Paracrine o Signalling cell release local mediators to target cells Synaptic Endocrine hormone release via bloodstream to target cell Cell signalling Intracellular signal transduction Extracellular signal molecule bind receptor on PM of target cell Intracellular signalling proteins (signalling cascade) Effector proteins o Metabolic enzyme Alter metabolism o Transcription regulatory protein Alter gene expression o Cytoskeletal protein Alter cell shape/movement Cells respond to combination of signals Different combination of signals direct the cell to perform different tasks/behave in different ways 4

5 Signalling molecule has different effects on different cell types I.e. ACh m3 receptor in heart pacemaker cells Rate of firing HR Salivary gland acini cells secretion Skeletal muscle cells contraction 3 major classes of membrane receptors 1. Ligand-gated ion channels (LICs)/Ionotropic receptors a. Allow passage of ions upon ligand binding (allosteric) b. I.e. GABAA receptor Cl- transmission inhibitory effect 2. G-protein coupled receptor (GPCR) a. 7 transmembrane (7TM) receptor activated by ligand/agonist active receptor + activated G-protein (3 subunits) GDP to GTP exchange on G-protein downstream effect (further intracellular signalling cascade) 3. Enzyme-linked receptors/catalytic receptors a. Dimer signalling molecule bind to extracellular binding domain b. Activate catalytic domain via a conformational change c. I.e. TLR, receptor tyrosine kinases, natriuretic peptide receptor (NPR) 5

6 Intracellular signalling A. Signalling by phosphorylation B. Signalling by GTP binding Signalling complex Preformed signalling complex on a scaffold protein o Inactive receptor + scaffold protein (with inactive intracellular signalling proteins) signal molecule receptor + scaffold protein ACTIVATED cascade of activated intracellular signalling proteins downstream signals Assembly of signalling complex on an activated receptor o Upon ligand binding to receptor inactive intracellular signalling proteins bind ACTIVATED receptor and ACTIVATED intracellular signalling proteins Downstream signals 6

7 Signalling complexes associated with membrane lipids o Assembly of signalling complex on phosphoinositide docking sites o Signal molecule activated receptor hyperphosphorylated phosphoinositides activated intracellular signalling proteins downstream signals Desensitisation 7

8 Lecture 3 (Signalling via G protein-coupled receptor) Describe the G protein cycle Explain how camp acts as a second messenger Understand the basics of GPCR structure Explain the main accessory proteins associated with GPCR signalling Concepts of receptor dimerisation and crosstalk Explain fluorescence and its use in microscopy Describe the use of GFP to label proteins Requirement of 2 nd messengers Low amount in resting state regulated synthesis/destruction Act through other proteins I.e. camp Adenylyl cyclase (AC) converts ATP camp AC need to be activated This explains low amount of camp in resting state (High [ATP] in cytoplasm) camp then activate KINASES (i.e. PKA) Adenylyl cyclase (AC) regulation via GPCR AC is the amplifier (activated by G-protein) GTP is required for the ligand-induced stimulation of AC (due to the high kinetic barrier of GDP-Gα exchange of GDP with GTP in order to activate G protein) GPCR structure 7 transmembrane α-helices (N-terminal extracellular/c-terminal cytosolic) Different classes of GPCR regulating a wide range of physiological events Activated by a huge range of ligands diversity of GPCR signalling 30-40% of marketed drugs target GPCRs o β-blockers, Angiotensin receptor blocker, histamine receptor blockers, opioid agonists Mutations in GPCR disease G protein Heterotrimeric (α, β, γ subunits) Each subunits of G protein act on various effector molecules 4 α subunits (i.e. CTX activate Gαs act on effect such as AC) Associated Heterotrimeric G protein is inactive (high kinetic energy barrier to GDP release) GEF (Guanine exchange factor) Kinetic barrier to GDP release is HIGH Replacement of GDP by GTP in the active site of Gα protein subunit TURN-ON signal o Require GEF, which happens to be the activated GPCR Heterotrimeric subunits dissociates into its 3 subunits The GPCR cycle 1) Ligand-induced conformational change in GPCR facilitates interaction w/ heterotrimeric G protein 2) Activated GPCR act as GEF for Gα subunit GDP GTP exchange TURN-ON signal 3) GTP-binding-induced separation of heterotrimeric G proteins 8

9 4) Interaction of G protein subunits with effectors 2 nd messenger activation signalling cascade a. GTP hydrolysis via GAP (GTPase activating proteins) 5) GPCR phosphorylation by GRKs (GPCR kinases) 6) Binding of β-arrestin to the phosphorylated GPCR a. Block further G protein-mediated signalling + target GPCR for internalisation 7) Reassociation of heterotrimeric G protein 8) GCPR internalisation followed by either: a. Degradation OR b. Recycling back to the PM 9) Attenuation of effector signalling through FB loops Desensitisation of GPCR and β-arrestin + GRKs After activation & signalling GPCR phosphorylation by GRK GRKs Phosphorylate the C-terminal conformational change exposing binding sites allow β-arrestin to bind Meanwhile, GAP GTP hydrolysis Reassociation of heterotrimeric G protein β-arrestin (phosphorylated residue on GPCR act as arrestin-binding sites) act as adaptors Desensitisation block further G protein-gpcr coupling (steric hindrance) Signalling attenuation via different FB loops 9

10 INTERNALISATION of GPCR via clathrin-coated pit endocytosis o Degradation o Recycling Receptor dimerisation Homodimerisation 2 same GPCR bound together by same ligand Oligomerisation Heterodimerisation 2 different class of GPCR bound together by the same ligand Specific ligand binding can dynamically regulate heterodimerisation Allosteric molecules can enhance/suppress downstream signalling Receptor dimerisation may play role in receptor maturation and correct trafficking Possible that dimerisation is a requisite for function Huge implications on drug designs Fluorescence Property of emitting EMR in the form of light as a result of the absorption of light from another source Fluorophore emits light after exposure to photon Light absorbed by an e- of the fluorophore gains energy and enter excited state (from ground state) As the fluorophore returns to the ground state emits photon at a longer wavelength (Stokes shift, emitted photon has less energy/frequency thus longer wavelength) o Emission of fluorescent light Dichroic mirrors SELECTIVELY pass light of a small range of wavelength but reflect light of other wavelength/colours 10

11 Using bioluminescence resonance energy transfer (BRET) to measure dimerisation 11